Our long-term goal is to understand how the vertebrate enteric nervous system (ENS) is specified and patterned during embryogenesis. Understanding the cellular and molecular processes involved in ENS formation will provide insights into how a complex structure such as a nervous system arises during development. Furthermore, our studies are of potential clinical importance in understanding the mechanisms and molecules that underlie pediatric conditions where the ENS fails to form correctly, such as Hirschsprung's disease (HSCR). The ENS is derived from the neural crest, a multipotent population of cells that migrates from the neural tube along specific pathways to generate a wide variety of neural and non-neural tissues. Different axial populations of neural crest cells contribute to specific structures and tissues. In this proposal we will address where, when, and how ENS precursors are specified during embryogenesis. Our studies use the zebrafish due to the model system's cell biological, molecular and genetic strengths that are uniquely appropriate for our proposed experiments. Specifically, we aim to answer the following questions: 1) Do all zebrafish enteric neural crest precursors arise from a distinct subset of vagal neural crest cells? We will determine the origin of the zebrafish ENS by lineage analysis. We will then investigate by cell ablation and cell transplantation the role of regulative interactions in specifying ENS precursor cell fate within the premigratory neural crest; 2) Which steps in enteric precursor development are affected by two mutations, lessen and enema? We have identified two zebrafish mutants that have significant reductions in the number of enteric neurons. We will characterize these mutants to determine cell biologically where, when, and how these mutations cause their ENS phenotypes; 3) What genes are affected in the lessen and enema mutants? We will identify the genes that are lesioned in lessen and enema and result in the ENS phenotype observed. Together the proposed aims will help increase our understanding of the mechanisms and molecules involved in generating the ENS and may provide valuable insights into the underlying causes of HSCR [unreadable] [unreadable]